The invention relates to assay methods which utilize unique labeling particles containing signal-generating moieties whose ratio can be adjusted. More specifically, the invention concerns particulate labels wherein the hue of the label can be adjusted incrementally.
It is often desirable to test a sample for reactivity against a multiplicity of reagents. For example, in tissue typing, the ability of cells in a particular tissue to immunoreact with a panel of antibodies raised with respect to marker antigens is required. Genetic testing often involves detecting the polymorphic form of a particular gene by assessing its hybridization to only one of many allelic possibilities. There are also techniques for sequencing by DNA hybridization performed by probing immobilized reference spots of simple sequence DNA with the fragment to be sequenced. In this technique, the ratios of binding of the fragment to be sequenced to each of the simple sequences is unique for each possible sequence in the target fragment. Proteins generated from cDNA libraries representing characteristic expression products of particular cells can be screened for reactivity with various targets. The targets may include antibodies, for example, or small molecule drug candidates.
In all of these cases, either repetitious multiple testing with a variety of reagents is necessary, or the reagents are displayed in an orderly array, such as on a microtiter plate or on a xe2x80x9cchipxe2x80x9d to assess the reactivity of a sample. In all of these procedures, the association of a particular label with formation of a complex by binding to a component of the sample requires identification of the label by its position in time (for sequential testing) or space (for testing against a panel) or by further measurements on the label, that require its release from the solid particles, e.g., by mass spectroscopy. For example, U.S. Pat. No. 5,721,099 describes solid supports used in multistage synthesis of combinatorial libraries. Labels attached to the solid support identify the structure of the member of the library attached. The reaction history of each particle can be determined by the release of the label and analysis to determine the nature and amount of the tag.
It would be advantageous if a sample could be simply assessed for its reactivity with a multiplicity of reagents without the necessity for physical or temporal separation. The present invention achieves this result by employing labels whose identity can be assessed in situ by their degree of xe2x80x9cgray,xe2x80x9d i.e., a property that can vary by small gradations between extremes over a wide range such as xe2x80x9cwhitexe2x80x9d to xe2x80x9cblack.xe2x80x9d While visible light is used as an illustration and as a means for explanation for simplicity herein, the invention includes embodiments of xe2x80x9ccolorxe2x80x9d which comprise other spectral properties such as radio frequency associated vibrational relaxation times in response to a magnetic field. Any property that can be varied over a wide range, as described above, can be employed. Thus, the nature of the label that is bound to a component of the sample is determinable by direct observation, enabling multiple parallel assays.
Attempts at multicolor labeling in a single sample have been made, most notably in xe2x80x9cpaintingxe2x80x9d chromosomes with labels of different color, a technique known as fluorescent in situ hybridization (FISH) with different colored labels for each chromosome. This technique has also been used to paint cells with different surface receptors by using binding ligands bearing different fluorophores. A summary of such approaches is set forth in Waggoner, A. et al., Human Pathology (1996) 27:494-502. The ability of the human eye to distinguish hues created by mixtures of different color components (although not with varied ratios of the same mixture of components) was demonstrated by Speel, E. J., et al., J Histochem Cytochem (1997) 45:1439-1446. Adaptation of this approach to oligonucleotide labels wherein dyes were applied at varying ratios to generate a number of different hues for the purpose of painting chromosomes was described by Morrison, L. E., et al., Cytometry (1997) 27:314-326. Although the labels are not particulate, and thus do not permit maximum signal intensity or the distinctive shape signal of uniform beads, this work does demonstrate that microscopic techniques can effectively detect and sort the hues generated when varying ratios of fluorophores are applied to a single label. As described in the catalog from Molecular Probes (Seattle), a microsphere of 100 nm diameter can be loaded with fluorescein to give an intensity equivalent to 7400 free fluorescein molecules with loading controllable to an accuracy better than plus or minus 5 percent.
The present invention offers the versatility of reagents and intensity of signal available through multihued beads wherein the particulate supports bearing signal-generating moieties are provided specific differentiable signals by virtue of these varying ratios. In addition, the labels can be evaluated not only in solution or suspension, but their location can be established through use of microscopic techniques in evaluating the samples.
Labels that can be identified by direct observation of their hue represent a great convenience in assays where multiple reactivities must be observed. The labels of the invention are particulate materials which contain at least two different signal-generating moietiesxe2x80x94for example, each providing light of a particular wavelength range either by reflectance or fluorescence. Thus, in one embodiment, an individual particle will contain moieties that emit or reflect light of different colors. By adjusting the relative amounts of the different moieties attached to the particle, the perceived xe2x80x9chuexe2x80x9d of the label will be different and it can be distinguished from other labels containing these moieties in different ratios. Effective use of these labels requires separate means for detection for each of the moieties coupled to the particle. Preferably, the particles contain at least three moieties which generate different wavelengths with a corresponding number of detection means.
Thus, in one aspect, the invention is directed to a label which comprises a particulate support to which is bound at least two signal-generating moieties, which moieties generate signals that can be distinguished in situ, such as light of different wavelengths. These labels are distinguishable by any instrumentation which contains separate means for detection for each of the at least two in situ signals generated. In general, separate detectors may be employed; however, a single detector may be employed using appropriate filters or other means, such as a prism or grating, to permit a single detector to perceive separately multiple signals, such as different wavelength ranges.
In particular, the in situ signals can be localized to obtain an overall pattern of labeling in a sample. Confocal optics and wide-field microscopy are able to distinguish structures on a microscopic scale and to permit observation of a geometric distribution of the labels of the invention.
In addition to establishing location of individual analytes on a microscopic level, the multihue particulate labels of the invention may be used macroscopically to map physiological phenomena. The labels can be used as xe2x80x9ctracersxe2x80x9d to follow endogenous pathways in complex systems. Thus, in another aspect, the invention is directed to a method to track physiological pathways by observing the distribution of the multihued labels of the invention, either as such, or provided with additional moieties showing an affinity for the pathway to be observed.
In another aspect, the invention is directed to an improved method for analyzing hue, or the ratio of signal-generating moieties on the multihued beads of the invention by observing the signals through wide-bandpass filters and correcting for the overlap.
In still another aspect, the invention is directed to a method to improve the definition of the hue associated with a particulate support by defining the observed signal in accordance with the known shape of the particulate support.
In another aspect, the invention is directed to a method to provide a desired ratio of signal-generating moieties on particulate support by coupling substantially all of the signal-generating moieties to be used to the support through linkers with differing lysis parameters. One embodiment of such linkers comprises short stretches of duplex DNA with the cleavage sites for different restriction enzymes for each different signal-generating moiety. For each single signal-generating moiety, more than one restriction site may be present. Alternatively, a single signal-generating moiety may be coupled through a multiplicity of linkers, each with a different cleavage parameter. Thus, a multiplicity of duplex DNAs would have different restriction enzyme sites. Under these conditions, the final ratio of signal-generating moieties can be controlled by varying the ratio of linkers used in fabricating the labeled particle and by the nature of the cleavage parameters used to treat the particulate.
In another aspect, the invention is directed to improved compositions of particulate supports which employ polymers that undergo phase transitions to open shapes such that the controlled variation in shape can be used to assist in adjusting the composition of labels on the particle.
In another aspect, the invention is directed to a collection of labels of the type described above wherein the ratio of the moieties differs from label to label in the collection. Typically, this collection of labels provides identifiable members that number at least twenty, preferably one hundred, more preferably five hundred, and still more preferably at least one thousand. Thus, if the reliability of detection of each color is plus or minus 10 percent, 10 gray labels exist for each signal and therefore 100 hues can be distinguished when two signal-generating moieties are included in each label. For use in analyzing a sample, each label will further be coupled to a different reagent.
In still another aspect, the invention is directed to a system for assessing the reactivity of a sample with a multiplicity of reagents coupled to labels, which system comprises the collection of labels bound to reagents described above along with separate detectors or their equivalents for the signals generated by each different moiety that is present on the particulates in the collection of labels.
Still another aspect of the invention relates to the use of collections of the labels in library versus library screening. Thus, for example, particles of different sizes may be associated with reagents that are potential opposite members of specific binding pairs such as antibodies/antigens where the antibody collection is bound to particulate supports of a different size as compared to the library of antigens. Successful binding can be detected by an aggregation of the smaller particles around the appropriately derivatized larger particle.
In still another aspect, particles of differing sizes can be used as a basis for homogeneous assays for analyte concentration in a competitive binding format or for affinity titrations by displacement of unlabeled analyte from larger particulates by competition from analyte analogs coupled to labeled, smaller beads.
In still another aspect, the invention is directed to methods of assessing the reactivity of components in a sample using the assay system described above.